The invention relates to transplantation of cells such as bone marrow cells.
Efforts to develop safe and effective therapies by transplanting various types of biological cells have been on the rise since the 1960s. For example, scientists began to consider organ transplantation, bone marrow transplantation, and enzyme supplementation to treat rare genetic disorders soon after the enzymatic defects in Gaucher""s and Niemann-Pick disease were discovered (see, e.g., Brady, New Engl. J. Med. 275:312, 1966). Today, certain organs (e.g., the kidney) are transplanted with great success and bone marrow transplantation is performed with increasing frequency, particularly in the context of treating cancer. Typically, cancer patients undergo autologous bone marrow transplantation; bone marrow cells are removed from the patient, maintained in an ex vivo culture while the patient is treated with radiation or by chemotherapy, and then transplanted back into the patient where they restore the bone marrow.
To effect genetic therapies, various cell types including bone marrow stromal cells (BMSCs) can be genetically modified and transplanted into a patient to treat a wide variety of disorders (see, e.g., U.S. Pat. No. 5,849,287).
The methods of the present invention are based on the discovery that certain adverse side effects (such as blood clotting and/or hemorrhage) caused by the infusion of transplanted cells, such as BMSCs, are due to the expression of tissue factor (TF) by the infused cells. BMSCs, which are described further below, are also described as mesenchymal stem cells (see, e.g., Prockop, Science 276:71, 1997). Accordingly, the methods of the invention are aimed at reducing the biological activity or level of TF in a patient, and can be carried out in a variety of ways. For example, one can: infuse fewer cells, e.g., BMSCs (or infuse the same number of cells over a longer period of time); reduce the expression or activity of TF (within the infused cells, e.g., by contacting the cells with a TF antagonist in vitro, or within the patient, e.g., by administering a TF antagonist to the patient; hamper the interaction of TF with factor VII(a); inhibit the activity of the TF-factor VII(a) complex once it has formed; or inhibit the coagulation cascade at any point downstream from formation of the complex (including inhibition of platelet activation). As implied by the foregoing, the term xe2x80x9cantagonistxe2x80x9d encompasses compounds (i.e., biological molecules, drugs, or other therapeutic agents) that interact with (and inhibit) TF directly or indirectly (by interacting with and inhibiting molecules that specifically bind TF (such as factor VII) or that lie downstream from the formation of the TF-factor VII complex). These means for reducing the biological activity or level of TF are described in detail below.
The methods of the invention may be practiced whenever patients are treated with cellular or gene therapies that employ transplanted cells, e.g., bone marrow cells, that express TF. For example, they can be practiced with cellular or gene therapies that employ bone marrow cells such as BMSCs.
The methods of the invention employ TF antagonists. Suitable antagonists include small molecules (i.e., molecules with a molecular weight below about 500), large molecules (i.e., molecules with a molecular weight above about 500), antibodies that specifically bind to and xe2x80x9cneutralizexe2x80x9d TF, and nucleic acid molecules that interfere with transcription or translation of TF (e.g., antisense nucleic acid molecules and ribozymes). TF antagonists also include compounds that interfere with the ability of TF to trigger blood clotting and/or coagulation. Preferably, a neutralizing antibody used in the present methods (or any of the other types of TF antagonists described herein) will neutralize at least 50%, more preferably at least 70%, and most preferably at least 80% (e.g., 85%, 90%, or 95%) of the activity of TF in a sample (e.g., a sample of BMSCs) containing TF. Antibodies and other types of antagonists are discussed in detail below.
In one embodiment, the new methods inhibit adverse vascular effects caused by introducing cells that express TF into a patient by reducing the total load of biologically active TF associated with the cells to less than about 25,000 ng/kg of the patient""s weight (e.g., less than about 10,000, less than about 5,000, or less than about 2,000 ng/kg of the patient""s weight).
The adverse effects that can be inhibited include hemorrhage, thrombosis, intravascular coagulation, disseminated intravascular coagulation, platelet aggregation, a reduced platelet count, reduced levels of the clotting factors V, VIII, and IX, an increased level of thrombin-antithrombin (TAT), an increased level of fibrin monomers, and an increased level of D-dimers. Preferably, these adverse effects are prevented entirely or inhibited sufficiently to prevent adverse symptoms. Thus, the new methods described herein can confer a benefit to the patient even if they only attenuate the adverse effects.
In some instances, the TF-expressing cells will be cultured prior to implantation into the patient. The cells can be cultured under the conditions described herein or under conditions known and used in the art.
The TF-expressing cells can be introduced into the patient (e.g., by infusion or injection into a blood vessel such as an artery or a vein, or by injection into a muscle, the dermis, or the peritoneal cavity) so that they come to reside in a desired location. The bone marrow cavities and intrarticular spaces are useful locations.
The TF-expressing cells can be introduced in a physiologically compatible solution, or can be implanted in a matrix (such as a gel, collagen, or bone-forming matrix, or a matrix composed of ceramic (e.g., a calcium phosphate ceramic), glass, or hydroxyapatite)) before they are introduced into the patient. Moreover, the cells can be wild-type TF-expressing cells or genetically modified TF-expressing cells. For example, the cells can contain a DNA construct encoding a coagulation factor such as factor VIII or factor IX or any of a wide variety of other biologically active polypeptides known to those of skill in the art. See for example, U.S. Pat. No. 5,849,287.
Patients having numerous types of diseases or disorders can benefit from the methods described herein. These methods may be particularly well suited for treating patients with hemophilia, cancer (such as breast cancer), or osteogenesis imperfecta.
As described further below, adverse vascular side effects can be inhibited by contacting TF-expressing cells (in vivo or ex vivo) with an oligonucleotide that inhibits transcription of a tissue factor gene, the stability of the tissue factor RNA, or the ability of tissue factor RNA to be translated into protein. The oligonucleotide can be a ribonucleic acid or a deoxyribonucleic acid molecule. Alternatively, the TF-expressing cells can be contacted with a ribozyme that cleaves tissue factor RNA. Where the antagonist of TF is an antibody, the antibody can be one that inhibits: the activity of tissue factor; a polypeptide that specifically binds to tissue factor; or a polypeptide that is active in the biochemical cascade that is initiated by the formation of a complex between tissue factor and factor VII. More specifically, the antibody can be TF1-E2, TF1-F7, HTF1-7B8, TF8-5G9, TF8-11D12, TF9-9C3, TF8-5G9, AP-1, 5G9, PW-4, 231-7, 12D10, hVII-B101/B1, hVII-D2/D4 or hVII-DC6/3D8 (each of which is further described below).
Antibodies useful in the methods of the present invention can be a monoclonal, polyclonal, or engineered antibodies that specifically bind to TF (as described more fully below). An antibody that xe2x80x9cspecifically bindsxe2x80x9d to a particular antigen, for example, to TF or to a polypeptide that interacts with (e.g., binds to) TF in the course of blood clotting, will not substantially recognize or bind to other molecules in a sample (i.e., a sample of biological material) that contains TF.
Given that an object of the present invention is to alter the expression or activity of TF in vivo (e.g., in BMSCs infused in the course of cell or gene therapy), a pharmaceutical composition containing, for example, an isolated nucleic acid molecule that is antisense to TF (i.e., that has a sequence that is the reverse and complement of a portion of the coding strand of a TF gene), or an antibody, small molecule, or other compound that specifically binds to TF is also a feature of the invention.
A xe2x80x9cpatientxe2x80x9d can be any animal including mammals such as humans and domesticated mammals, e.g., dogs, cats, horses, cows, sheep, and pigs.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
The preferred methods and materials are described below in terms that are meant to illustrate, not limit, the invention. One of ordinary skill in the art would recognize methods and materials that are similar or equivalent to those described herein, and that can be used in the practice or testing of the present invention.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.